PROJECT SUMMARY The group A Streptococcus (GAS, S. pyogenes) causes ~700 million human infections annually. Perhaps the most important regulatory proteins in this bacterium are the control of virulence regulator (CovR) and sensor (CovS), which together form a two-component system (TCS). CovR/S potentiate the ability of GAS to cause non-invasive infections (e.g. pharyngitis), but this activity comes at the expense of invasive infection (e.g. necrotizing fasciitis) virulence. Importantly, covR or covS mutant strains are selected for during invasive GAS infections, creating hyper-virulent isolates. Despite the importance of CovR/S to GAS pathogenesis there remain significant gaps in our knowledge: (a) almost two decades have passed since the finding that covR and covS mutant strains are not equivalent, with the most striking difference being the levels of mRNA encoding for the secreted protease SpeB (a several thousand-fold difference); (b) the accessory protein regulator of cov (RocA) is a major activator of CovR activity, but the mechanism by which it does this is unknown; and (c) while rocA mutants can be recovered in a strain-specific manner from invasive infections, similar to the isolation of covR or covS mutants, a unique aspect of rocA is that certain GAS serotypes are exclusively rocA mutant, consistent with the regulatory and disease-phenotype consequences of losing RocA activity being distinct from losing CovR or CovS activity. The goals of this proposal are to provide a molecular explanation for why we observe regulatory disparity between these naturally-occurring GAS derivatives, to identify the virulence consequences of this disparity, and to determine how the regulatory components interact. In Aim 1 we will delineate physical and functional interactions between RocA and CovS. Preliminary data are consistent with the hypothesis that RocA complexes with CovS to enhance CovS kinase activity towards CovR. We will test the requirement of CovS kinase and phosphatase activities for RocA-mediated regulation after creating covS mutant GAS strains deficient in individual activities. We will also use two approaches to identify interactions between RocA and the CovS or CovR proteins. In Aim 2 we will determine the virulence characteristics of parental and naturally-occurring GAS variants, and the molecular basis of observed regulatory disparity between these isolates. Genome-wide approaches will be used to test the hypothesis that CovR~P and non-phosphorylated CovR have repressor activity, and that they target different subsets of CovR/S-repressed genes. To characterize how some GAS serotypes can tolerate being exclusively rocA mutant we will use ex vivo and in vivo models of infection, testing the hypothesis that rocA mutation, but not covR or covS mutation, only minimally impacts the ability of GAS to cause upper respiratory tract infections. Completing the proposed research will generate insights into why GAS variants are recovered in clinically- significant numbers, will generate the first mechanistic data for a TCS-regulating accessory protein from a Lactobacillale pathogen, and will answer a long-standing question in the field of streptococcal gene regulation.